Magnitude of Mycobacterium tuberculosis, drug resistance and associated factors among presumptive tuberculosis patients at St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia

Background Mycobacterium tuberculosis (M. tuberculosis) remains one of the most significant causes of death and a major public health problem in the community. As a result, the aim of this study was to determine magnitude of Mycobacterium tuberculosis, its drug resistance, and associated factors among presumptive tuberculosis (TB) patients at St. Paul’s Hospital Millennium Medical College, Addis Ababa, Ethiopia. Methods Cross-sectional study was conducted at St. Paul’s Hospital Millennium Medical College (SPHMMC), Addis Ababa, Ethiopia from January to July 2019. Demographic and clinical data were collected by structured questionnaire through face to face interview. Using microscopic examination and GeneXpert MTB/RIF assay and culturing in the Lowenstein-Jensen (LJ) culture media, we collected and analyzed both pulmonary and extra-pulmonary clinical samples. Data were analyzed by SPSS version 23. Binary logistic regression was done to identify the associated risk factors and p-value less than 0.05 was taken as significant association. Results Of the total 436 respondents, 223(51%) were male. The mean ±SD age of the participants was 38±17years. Overall, 27/436(6.2%) of the participants had confirmed Mycobacterium tuberculosis using the GeneXpert MTB/RIF assay and LJ culture media, and two isolates were resistant to RIF and one to INH medication, with two (0.5%) being MDR-TB. MTB infection was associated with previous TB contact history, patient weight loss, and CD4+ T-cell counts of 200-350/mm3 of blood. Conclusion The magnitude of M. tuberculosis and MDR-TB in this study underscores the need for improved early case detection and management of MDR-TB in order to reduce transmission and patient suffering.


Methods:
A cross-sectional study was conducted at SPHMMC, Addis Ababa, Ethiopia from Jan to July 2019. Socio-demographic data were collected by using structured questionnaire in face to face interview with patients. Sputum samples were collected and the laboratory analysis was done by microscopy and X-pert MTB/RIF assay and LJ culture media. Data were analyzed by SPSS version 23. Binary logistic regression was done to identify the associated risk factors, and p-value less than 0.05 was taken as significant association.

Results:
Of the total of 436 respondents, 223 (51%) were male. The mean ±SD age the participants were 38±17years. Out of the total participants, the overall confirmed Mycobacterium tuberculosis was through X-pert MTB/RIF assay and LJ culture media was 27 (6.2%), and three isolates were resistant for either INH or RIF drug, while two of them were MDR-TB based on line probe assays method. Previous TB-contact history, patient weight loss, having pneumonia with chest X-ray finding, and CD4 + T-cells count 200-350/mm 3 of blood were significantly associated predictors for MTB infection.

Background
Tuberculosis (TB) is an infectious disease caused by strains belonging to the Mycobacterium tuberculosis complex. It is transmitted by respiratory route when a patient is coughing or sneezing, and one strain of TB, Mycobacterium bovis, can be caused by drinking not boiled milk [1]. World Health Organization (WHO) estimated that 10 million people developed tuberculosis (TB) and 1.6 million died of TB globally in 2017 and one-fourth of people infected with latent Mycobacterium tuberculosis [2].
Globally, the estimated prevalence of MDR-TB was 3.3% in newly diagnosed patients in the WHO 2015 report. This was higher to 20% in patients with a history of anti-TB treatment(30).
A global TB report estimated that there were about 230,000 (247 per 100,000 population) incident cases of TB in Ethiopia.
Ethiopia ranks seventh among the world's 22 high-TB-burden countries, 10th among high-TBpandemic countries, and fourth in sub-Saharan Africa [3].
Based on the 2005 nationwide survey in Ethiopia, the prevalence of MDR-TB was 1.6% among new cases and 11.8% in the retreatment cases and rifampicin resistant was lower than 2% in new cases [4].
High mortality rate was observed in different health institution of the Northern Ethiopia; 87 (11.3%) patients died in Mekelle Hospital and Ayder Comprehensive Hospital [5], 38 (14.02%) children from TB/HIV co-infected University of Gondar Comprehensive Specialized Hospital [6] and from multidrug resistant tuberculosis (MDR-TB) data showed that 61(29.47%) of the patients died in different hospitals of Amhara region, Northwest Ethiopia [7]. Generally in Ethiopia, TB mortality rate declined from 393.8/100,000 to 100/100,000 between 1990 and 2016 (with a total decline of 75%), which indicates slow decline and resulted males had higher TB mortality rate than females [8].
Sputum smear microscopy remains the most common way to diagnose pulmonary TB.
Depending on the report and method used, smear microscopy can accurately detect TB in 20% to 80% (using fluorescence microscopy methods) of TB cases. However, it could be used to diagnose TB when sputum has sufficient bacillary load, and it cannot detect drug resistance.
Thus, HIV-associated TB often goes undetected because people living with HIV (PLHIV), especially those with severe immunosuppression generally have very low numbers of bacilli [9].
Hence, X-Pert used as an initial diagnostic test for TB detection and rifampicin resistance detection in patients suspected of having TB, MDR-TB, or HIV-associated TB is sensitive and specific [10]. Therefore, the goal of this study was to determine magnitude of Mycobacterium tuberculosis and its associated factors among TB-presumptive patients referred to St. Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia.

Study area
The study was conducted in St Paul's Hospital Millennium Medical College, Addis Ababa, Ethiopia. It is currently has 392 beds, with an annual average of 200,000 patients and a catchment population of more than 5 million. The hospital receives referrals from around the country and is under the guidance of the Ethiopian Federal Ministry of Health.

Study design and period
A cross-sectional study was conducted at SPHMMC, Addis Ababa, Ethiopia from Jan to July 2019, and all patients who visited were source population while all Mycobacterium tuberculosis 5 presumptive patients visited microbiology laboratory and fulfill the inclusion criteria were considered as study population.

Inclusion and Exclusion criteria
All presumptive Mycobacterium tuberculosis patients visiting Microbiology laboratory were included and patients who have inadequate specimen, previous history of known multidrug resistance for Mycobacterium tuberculosis were excluded from the study.

Variables
Magnitude of Mycobacterium tuberculosis and its drug resistance pattern among presumptive patients dependent variables. Whereas, socio-demographic characteristics (age, sex, etc), possible risk factors like; TB contact history, previous treatment for TB, presumptive DRTB, BCG vaccination status, CD4 and HIV viral load counts were independent variables.

Sample size and sampling technique
The sample size was estimated based on the assumption of single population proportion formula, considering the previous study conducted in Debremarkose, Northwest Ethiopia taken as 23%, 5% marginal error, and 95% confidence level to get the highest sample size, the calculation result determined as: Where: n = minimum sample size, P = estimated proportion of Mycobacterium tuberculosis for the study population, and taking 10% non-response rate, the final sample size become 422 participants.
d= the margin of sample error, z α /2= the standard normal variable at 1-α /2 confidence level and we used consecutive sampling technique was used to select the study population.

Data collection procedure
Data collectors were trained and informed how to collect the data. Structured questionnaire was used to collect the socio-demographic status and associated risk factors of the study participants.
From each presumptive Mycobacterium tuberculosis patients, 2-4 ml of clinical sputum sample was collected.

Laboratory procedures
Microscopy and gene X-pert® MTB/RIF were done.
The Lowenstein-Jensen (LJ) medium was used which incorporates congo red and malachite green to inhibit unwanted bacteria for culturing. Once good growth was obtained, the positive slants were stored in a cool, dark place to archive the positive M. tuberculosis isolates. 7

Data Quality Assurance
The questionnaire was pre-tested and proper training prior to the actual data collection was given for data collectors. The necessary adjustments were made after the pre-test. The quality of data was maintained through strictly following the pre-analytical, analytical and post-analytical steps.

Data analysis and interpretation
The collected data were entered to EPI info 2002 version 3.32 after data editing and cleaning it was exported to SPSS version 23 windows software computer program for analysis. The logistic regression was employed to assess the association between of different factors. A p-value of less than 0.05 was considered as statistical significance.

Socio-demographic characteristics
The total of 436 respondents were included in the study, of this 223 (51%) were male. The mean ± SD age the participants were 38±17years.

Magnitude of M. tuberculosis and resistance pattern
Out of the total participants, 36(8.3%) were detected with X-pert, and of this figure only 2 (0.5 %) of them were RIF resistant. Regarding culture result, 27(6.2%) was positive and one M.

Bivariate analysis
Presence of contact history with tuberculosis infected patients, pneumonia confirmed with chest-X-ray examination, and CD4 + results were associated factors for M. Tuberculosis in the bivariate logistic analysis, however, none of the factors associated in multivariable analysis, table 4.

Discussion
The highest TB frequency was observed in age groups of 35-49 years old, living in 4-6 family size / house, regarding to occupation; laborer workers, having monthly income 1001-2000 Ethiopian Birr. The TB magnitude among this productive age group (35-49) years of study participants was (9 [33.3%]). This might be due to more exposure to the high workload, and wide range of mobility in these age-groups.
In this study it seems that as the number of family size per house hold increase the prevalence of smear positivity also increases. Family size 5-6 was highly affected by Mycobacterium tuberculosis. Different studies indicated individuals living in larger family size members and malnutrition are at higher risk of developing pulmonary tuberculosis [12], however our study revealed that no association family size/house hold and Mycobacterium tuberculosis. The distribution of pulmonary tuberculosis was also measured in terms of contact history with chronic coughers, smoking habit and alcoholism to trace the epidemiological features of the disease. In this study, the magnitude of pulmonary tuberculosis was not significantly high in those who had contact with TB infected patients, previous history of anti-TB treatment, drinking of alcohol and in those who were smokers. These findings were different from the studies done in Addis Ababa, Ethiopia in 2011 and north Gondar in 2015 [13,14].
The possible reason might be due to lower number of participant diagnosis the reason for presumptive DRTB in our cases and using more number of participants from urban resident.
Higher result again observed in previously treated patients 20/436 (4.5%) with anti-TB drugs and in new patients for presumptive drug resistance tuberculosis 24/436 (5.5%).
Statistically significant association was observed between culture positive pulmonary tuberculosis and TB contact History and some of tuberculosis patient symptoms weight loss, having pneumonia and CD 4 + counts. The previous study also indicated that pulmonary tuberculosis associated with the level of CD4 + in HIV patients and the amount of virus present in the participant's blood [13,15]. from Metehara sugar factory hospital, eastern Ethiopia (14.2%) 18 and 124 (32.2 %) of studied in two public hospitals in East Gojjam zone, northwest Ethiopia [19].
As compared to retrospective study report, from the University of Gondar Hospital from January 2013 to August 2015, prevalence of (24.6%), we found low result [20].
Our finding also lower than 23.2% of the study conducted in Debre markos Referral Hospital, Ethiopia using Gene X-pert MTB/RIF assay.
The possible reason for the difference might be associated with the variation of the diagnostic methods we used, for example in our cases we used sputum sedimentation concentration technique for microscopic smear examination, Gene X-pert assay and finally LJ culture for confirmation whereas, a single diagnostic tool used in the previous study like; stained by Ziehl- The bivariate logistic analysis showed that presumptive drug resistance tuberculosis two times more likely (2.6 (95% CI 0.6, 12, p=0.2)) to develop tuberculosis than presumptive tuberculosis; also having the symptoms of night sweating two times more likely (2.4(95% CI 0.8, 7.2, p=0.1)) to develop tuberculosis than those who did not the symptoms of night sweating. Having the presence of chest pain also (1.6 (95% CI 0.8, 3.7, p=0.2)) times more likely to develop Mycobacterium tuberculosis than from those who did not have chest pain.

Conclusion
Presence of contact history with previous tuberculosis infected patients, patient weight loss, presence of pneumonia with radiological examination, and CD4 + results were the identified symptoms and factors associated from M. Tuberculosis in the bivariate logistic analysis.
In general, this study highlights low magnitude Mycobacterium tuberculosis among presumptive patients visited to SPHMMC, Addis Ababa, Ethiopia, however from the total of three strains, two of MDR strains were observed on those who have history of failure, relapse and previously treated with anti-TB treatment.
Health education about tuberculosis, TB control programs should be continued and large community based study also recommended to sustain this low result of the disease. 13 Strengthening TB infection control activities and proper implementation of DOTS are also recommended to reduce the burden of MDR-TB.

Ethics approval and consent to participate
The proposed study was approved by the Department of Medical Laboratory Science, Addis Ababa university research and ethics committee concerning the ethical issues giving a reference number SR/LS/025/19.

Consent for publication
Not applicable. This study does not contain any individual or personal data.

Availability of data and materials
All data relevant to this study are available on the manuscript.

Funding
This research work was supported by Addis Ababa University, Ethiopia. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. List of tables